A Luciferase Fusion Library of High Throughput Profiling of Protein Stability

描述（由申请人提供）：泛素化-蛋白酶体途径的靶向蛋白质降解在包括癌症生物学在内的许多生物学领域发挥着重要作用。蛋白质泛素化是由一组三种酶（E1泛素激活酶E1， E2泛素结合酶E2泛素蛋白连接酶）进行的，它们将泛素转移到目标蛋白上，发出信号，由26S蛋白酶体破坏。许多肿瘤抑制基因和癌基因编码E3连接酶（包括pVHL、FBW7、MDM2、BRCA1、Park2和E6）或泛素化靶点（包括p53、E2F-1、Cyclins、c-Myc和c-Jun）。此外，所有与癌症相关的E3连接酶的潜在底物从未以高通量的方式系统地研究过。建立一个完整的人类基因组编码的所有E3连接酶的潜在底物列表，不仅将促进我们对生物学的理解，而且有助于开发更有效的治疗方法来治疗由异常靶向蛋白降解引起的人类疾病。众所周知，当不稳定的泛素化靶标与生物发光报告因子（如萤火虫荧光素酶）融合时，可以促进其融合伙伴的降解。因此，测量一个蛋白- x萤火虫荧光素酶融合蛋白的报告活性可以作为监测内源性蛋白- x在响应给定信号时蛋白稳定性变化的有效手段。为此，我开发了一种高通量的双荧光素酶报告法来测量蛋白质的稳定性。本提案的目的是通过生成具有内置reniall荧光素酶控制的人类开放阅读框（ORF）萤火虫荧光素酶融合克隆文库，并进行原理验证实验，将该方法的应用扩展到全基因组规模，以建立该文库用于筛选涉及癌症或其他人类疾病的E3连接酶的底物和/或生物标记物的有效性。

DESCRIPTION (provided by applicant): Targeted protein degradation via ubiquitination-proteasome pathway plays an important role in many areas of biology including cancer biology. Protein ubiquitination is carried out by a set of three enzymes (an E1 ubiquitin-activating enzyme E1, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin protein ligase), which transfers the ubiquitin to the target protein to signal for destruction by the 26S proteasome. A number of tumor suppressor genes and oncogenes encode either E3 ligases (including pVHL, FBW7, MDM2, BRCA1, Park2 and E6) or ubiquitination targets (including p53, E2F-1, the Cyclins, c-Myc and c-Jun). Moreover, the potential substrates of all cancer-related E3 ligases have never systematically investigated in a high throughput manner. Establishing a complete list of potential substrates of all E3 ligases encoded by human genome will not only advance our understanding of biology but also help develop more effective therapeutics for treating human diseases caused by aberrant targeted protein degradation. It is well established that unstable ubiquitination targets, when fused with a bioluminescence reporter (such as firefly luciferase), can promote the degradation of their fusion partners. Therefore, measuring the reporter activity of a Protein-X firefly luciferase fusion protein could serve as an effective means to monitor the change of protein stability of endogenous Protein-X in response to a given signal. To this end, I have developed a high throughput, dual- luciferase reporter assay for measuring protein stability. The objective of this proposal is to extend the application of this approach to a genome-wide scale by generating a library of human open reading frame (ORF) firefly luciferase fusion clones with built-in reniall luciferase control and performing proof-of-principle experiments to establish the utility of this library for screening substrates and/or biomarkers of E3 ligases involved in cancer or other human diseases. PUBLIC HEALTH RELEVANCE: Ubiquitin-dependent protein degradation is involved in many aspects of cancer research since many tumor suppressor genes and oncogenes encode ubiquitination enzymes and substrates. The goal of this research proposal is to establish a high throughput screening approach for measuring protein stability genome-widely and to test the utility of this approach to identify potential substrates of cancer-related ubiquitination enzymes. Therefore, it holds great promise to facilitate the development of effective therapeutic cures for treating cancer and other human diseases caused by aberrant ubiquitin-dependent protein degradation.